Project Summary The pylorus is the muscular junction between the stomach and intestine that regulates the passage of food into the duodenum. A relatively common postnatal pathology in infants is hypertrophic pyloric stenosis (HPS), which presents with projectile vomiting and inability to feed. In adults, pyloric malfunction can lead to gastro- duodenal reflux. The molecular and cellular etiologies underlying these pyloric pathologies are unknown, but a better understanding of the development of the pylorus and its associated muscular connections may shed light on these mechanisms. The work proposed in this application is based on a recent microarray analysis that identified a number of genes expressed uniquely at the pylorus, including the transcription factors, Nkx2-5 and Gata3. Preliminary data indicate that Gata3 and Nkx2-5 are co-expressed in smooth muscle cells within a unique domain at the pylorus. Both proteins are also expressed in previously undescribed superficial cord-like structures composed of smooth muscle cells that connect the esophagus to the pyloric region. In Gata3 null mice, specific groups of smooth muscle cells are lost (including the muscular cords), resulting in reduced pyloric constriction and alterations in the shape of the antrum. Previous studies in the chick model indicate that Nkx2-5 loss also affects pyloric constriction, though its effects at the cellular level were not studied. The hypothesis driving this work is that Gata3 is required for development of functionally relevant smooth muscle populations of the pylorus and is itself regulated by Nkx2-5. In Aim 1 of these studies, a careful analysis of pyloric development will be carried out, integrating cellular, molecular and functional aspects of pyloric development in the wild type mouse and setting an important baseline for further studies in vertebrate models. The functional role of the smooth muscle cords will also be studied. In Aim 2, the functional interaction between Gata3 and Nkx2-5 will be investigated and the epistatic relationship between these two proteins will be clarified. In the course of these studies, a novel tissue culture system will be employed to analyze the development of pyloric contractile function and the effects of loss of Nkx2-5 or Gata3 on this function. Together, the work described in this proposal will improve our understanding of the development as well as the function of specific smooth muscle groups at the pylorus and clarify the role of Gata3 and Nkx2-5 in pyloric development, information that could ultimately impact the clinical management of pyloric pathologies in humans.